Submitted 28 July 2015Accepted 27 December 2015Published 2 February 2016

Corresponding authorAndrea Gori agorimailgmailcom

Academic editorMoacutenica Medina

Additional Information andDeclarations can be found onpage 10

DOI 107717peerj1606

Copyright2016 Gori et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Physiological response of the cold-watercoral Desmophyllum dianthus to thermalstress and ocean acidificationAndrea Gori12 Christine Ferrier-Pagegraves2 Sebastian J Hennige1 Fiona Murray1Ceacutecile Rottier2 Laura C Wicks1 and J Murray Roberts1

1Centre for Marine Biodiversity and Biotechnology Heriot-Watt University Edinburgh ScotlandUnited Kingdom

2Coral Ecophysiology Centre Scientifique de Monaco Monaco Principality of Monaco

ABSTRACTRising temperatures and ocean acidification driven by anthropogenic carbonemissions threaten both tropical and temperate corals However the synergisticeffect of these stressors on coral physiology is still poorly understood in particularfor cold-water corals This study assessed changes in key physiological parameters(calcification respiration and ammonium excretion) of the widespread cold-watercoral Desmophyllum dianthusmaintained forsim8 months at two temperatures(ambient 12 C and elevated 15 C) and two pCO2 conditions (ambient 390 ppmand elevated 750 ppm) At ambient temperatures no change in instantaneouscalcification respiration or ammonium excretion rates was observed at either pCO2levels Conversely elevated temperature (15 C) significantly reduced calcificationrates and combined elevated temperature and pCO2 significantly reduced respirationrates Changes in the ratio of respired oxygen to excreted nitrogen (ON) whichprovides information on the main sources of energy being metabolized indicateda shift from mixed use of protein and carbohydratelipid as metabolic substratesunder control conditions to less efficient protein-dominated catabolism underboth stressors Overall this study shows that the physiology of D dianthus is moresensitive to thermal than pCO2 stress and that the predicted combination of risingtemperatures and ocean acidification in the coming decades may severely impact thiscold-water coral species

Subjects Ecology Marine BiologyKeywords Cold-water corals Thermal stress Ocean acidification Coral calcificationCoral respiration Coral excretion

INTRODUCTIONIncreases in anthropogenic carbon emissions leading to rising sea temperatures andocean acidification have resulted in extensive tropical coral bleaching (eg Hoegh-Guldberg 1999Mcleod et al 2013) and decreased coral calcification rates (eg Gattusoet al 1998 Chan amp Connolly 2012Movilla et al 2012 Bramanti et al 2013) The com-bination of rising temperatures and ocean acidification are substantial threats for coralsin the next few decades (Hoegh-Guldberg et al 2007 Silverman et al 2009 Erez et al

How to cite this article Gori et al (2016) Physiological response of the cold-water coral Desmophyllum dianthus to thermal stress andocean acidification PeerJ 4e1606 DOI 107717peerj1606

2011) While considerable research efforts have focused on tropical and temperate coralsless is known about the effects of ocean warming and acidification on cold-water corals(CWC) (eg Guinotte et al 2006 Rodolfo-Metalpa et al 2015 and references therein)These corals are among the most important ecosystem engineering species (sensu JonesLawton amp Shachak 1994) in the deep sea where they build three-dimensional frameworks(Roberts Wheeler amp Freiwald 2006) that support a highly diverse associated fauna (Henryamp Roberts 2007 Buhl-Mortensen et al 2010) Scleractininan CWC are most commonlydistributed at temperatures between 4 C and 12 C (Roberts Wheeler amp Freiwald2006) and show species-specific responses to temperatures above their natural thermalrange For instance elevated seawater temperatures increased calcification in the non-reef forming Dendrophyllia cornigera (Naumann Orejas amp Ferrier-Pagegraves 2013 Gori etal 2014a) had no effect on calcification in the solitary coral Desmophyllum dianthus(Naumann Orejas amp Ferrier-Pagegraves 2013) and had either no effect on the reef-formingLophelia pertusa calcification (Hennige et al 2015) or induced mortality (Brooke et al2013) depending upon the site of origin and change in temperature

In comparison to thermal stress CWC seem to have a general capacity to withstandocean acidification under experimental time periods of up to 12 months Decreases inpH did not affect calcification rates in both the reef forming L pertusa andMadreporaoculata (Form amp Riebesell 2012McCulloch et al 2012Maier et al 2012Maier et al2013a Hennige et al 2014 Hennige et al 2015Movilla et al 2014a) or the non-reefforming D cornigera D dianthus (Movilla et al 2014b Rodolfo-Metalpa et al 2015)Caryophyllia smithii (Rodolfo-Metalpa et al 2015) or Enallopsammia rostrata (McCullochet al 2012) However whether calcification can be sustained indefinitely remains unclearas seawater acidification has been shown to affect coral metabolism (Hennige et al 2014)increasing energy demand (McCulloch et al 2012) and leading to up-regulation of genesrelated to stress and immune responses energy production and calcification (Carreiro-Silva et al 2014) Coral responses to ocean acidification may also depend on seawatertemperature (eg Reynaud et al 2003 Edmunds Brown amp Moriarty 2012) and evidenceis now emerging that only when these two factors are combined (as is likely with futureclimatic changes) do the real effects of ocean change become apparent (Reynaud et al2003 Roberts amp Cairns 2014)

This study focused on the combined effects of increased temperature and pCO2 onkey physiological processes of the cosmopolitan solitary CWC D dianthus (Cairns ampZibrowius 1997) sampled in the deep waters of the Mediterranean Sea Calcificationrespiration and ammonium excretion were quantified in corals maintained oversim8months under a combination of conditions that replicated ambient temperature andpCO2 levels (12 Cmdash390 ppmMovilla et al 2014b) and elevated temperature andpCO2 levels predicted in the IPCC IS92a emission scenarios (15 Cmdash750 ppm followingRiebesell et al 2010) We hypothesize that the combination of elevated temperaturesand pCO2 will have a greater impact on coral calcification respiration and excretionthan single stressors Analysis of the ratio of respired oxygen to excreted nitrogen (ON)which is a physiological index providing information on the main sources of energy beingmetabolized (Sabourin amp Stickle 1981 Yang et al 2006 Zonghe et al 2013) was used to

Gori et al (2016) PeerJ DOI 107717peerj1606 216

Figure 1 The cold-water coralDesmophyllum dianthus Photo by A Gori

reveal whether corals are mainly metabolizing proteins carbohydrates or lipids giving afurther indication of coral stress under the experimental conditions

MATERIALS AND METHODSCoral collection and maintenanceSpecimens of D dianthus (Esper 1794) (Fig 1) were collected in the Bari Canyon (Adri-atic Sea Mediterranean Sea 41172622primeN 17166285primeE 430 m depth) by the Achille M4and Pollux III ROVs and kept alive on board the RV lsquoUraniarsquo during the cruise ARCA-DIA (March 2010) Corals were transported to the Centre Scientifique de Monaco (CSMMonaco Principality of Monaco CITES permit 2012MC7725) and maintained there forsim35 months in 50 L continuous flow-through tanks with seawater pumped from 50 mdepth at a rate of 20 L hminus1 Water temperature was maintained close to in situ conditions(12plusmn 10 C) and powerheads provided continuous water movement within the tanksCorals were fed five times a week with frozenMysis (Crustacea Eumalacostraca) andadult Artemia salina (Crustacea Sarsostraca) For experimental work 12 specimensof D dianthus were transferred to Heriot-Watt University (Edinburgh Scotland UKCITES permit 2012MC7929) and kept under collection site ambient conditions forsim2 months before beginning the experimental incubations Corals were then placedinto ambient temperature and pCO2 (12 Cmdash390 ppm) levels and predicted futureconditions following the IPCC IS92a emission scenarios (Riebesell et al 2010) ambienttemperature and elevated pCO2 (12 Cmdash750 ppm) elevated temperature and ambientpCO2 (15 Cmdash390 ppm) and elevated temperature and pCO2 (15 Cmdash750 ppm)

Gori et al (2016) PeerJ DOI 107717peerj1606 316

For each treatment there were three replicate systems ofsim80 L tanks holding onecoral each The tanks were equipped with pumps and filtration units to ensure adequatewater mixing and filtration Tanks were closed systems filled with seawater collectedfrom the east coast of Scotland (St Andrews) with partial water changes (20) every twoweeks Ambient and mixed elevated pCO2 air mixes were bubbled directly into the tanksas described by Hennige et al (2015) Gas mixing was achieved to target levels by mixingpure CO2 with air plumbed from outside of the laboratory building in mixing vesselsMixed or ambient gas was then supplied to appropriate experimental systems Targetgas levels were checked and adjusted daily using a LI-COR 820 gas analyzer calibratedusing pre-mixed 0 and 750 CO2 ppm gases (StG gases) All replicate systems werehoused in darkness within a temperature-controlled room at 9 Cplusmn 05 C and watertemperatures in the systems (12 Cplusmn 05 C and 15 Cplusmn 05 C) were controlled throughAqua Medic T-computers and titanium heaters (Aqua Medic TH-100) Experimentalsystem temperature salinity (YSI 30 SCT) and pH(NBS) (Hach HQ 30D) were measuredand recorded throughout the duration of experiment Average pH(NBS) (plusmnstandarddeviation) values for each treatment (pooled between 3 replicate tanks) over this 8 monthperiod were 12 Cmdash380 ppm= 796plusmn 006 12 Cmdash750 ppm= 792plusmn 006 15 Cmdash380 ppm= 797plusmn 004 and 15 Cmdash750 ppm= 790plusmn 006 Further details about theincubation systems are available in Hennige et al (2015) which support routine pH(NBS)measurements and highlight the stability of these systems over prolonged time periods(Table S1) Corals were fed 3 times a week with a controlled supply of 2 krill (Gammafrozen blister packs) per polyp per feeding event

Physiological measurementsAfter 236 days under experimental conditions four sets of incubations were performedone for each experimental condition to assess rates of calcification respiration andammonium excretion Each incubation started with the preparation of 1 L of 50 microm pre-filtered seawater 140 ml of this seawater was sampled for the initial determination of thetotal alkalinity (TA) (120 ml) and ammonium concentration (20 ml) as described belowThe remaining filtered water was equally distributed between 4 incubation chambers(200 ml each) One chamber was left without a coral polyp and used as a control Threeother chambers housed one polyp each from a different replicate system Polyps wereincubated for six hours in the individual chambers that were completely filled (withoutany air space) and hermetically closed according to the standardized protocol developedby Naumann et al (2011) Constant water movement inside the beakers was ensured by ateflon-coated magnetic stirrer At the end of the incubation 140 ml of seawater was takenfrom each incubation chamber and split between storage vessels for the determination ofthe final TA and ammonium concentration as described below

Coral calcification rates were assessed using the alkalinity anomaly technique (Smithamp Key 1975 Langdon Gattuso amp Andersson 2010) assuming a consumption of 2moles of alkalinity for every mole of calcium carbonate produced (Langdon Gattusoamp Andersson 2010) Seawater samples (120 ml) from before and after incubation weresterile filtered (02 microm) and fixed with HgCl2 to prevent further biological activity TA

Gori et al (2016) PeerJ DOI 107717peerj1606 416

was determined on 6 subsamples of 20 ml from each chamber using a titration systemcomposed of a 20 ml open thermostated titration cell a pH electrode calibrated onthe National Bureau of Standards scale and a computer-driven titrator (Metrohm 888Titrando Riverview FL USA) Seawater samples were kept at a constant temperature(250plusmn 02 C) and weighed (Mettler AT 261 LrsquoHospitalet de Llobregat Spain precision01 mg) before titration to determine their exact volume from temperature and salinityTA was calculated from the Gran function applied to pH variations from 42 to 30 as thefunction of added volume of HCl (01 mol Lminus1) and corrected for changes in ammoniumconcentration resulting from metabolic waste products (Jacques amp Pilson 1980 Naumannet al 2011) Change in the TA measured from the control chamber was subtracted fromthe change in TA in the chambers with corals and calcification rates were derived fromthe depletion of TA over the 6 h incubation

Respiration rates were assessed by measuring oxygen concentration in the incubationchambers during incubations with optodes (OXY-4 micro PreSens Germany) calibratedusing sodium sulfite and air saturated water as 0 and 100 oxygen saturation valuesrespectively Variations in oxygen concentrations measured from the control chamberwere subtracted from those measured in the coral chambers and respiration rates werederived from the recorded depletion of dissolved oxygen over the incubation Oxygenconsumption rates were converted to C equivalents (micromol) according to the equationC respired =O2 consumed middot RQ where RQ is a coral-specific respiratory quotient equalto 08 mol Cmol O2 (Muscatine McCloskey amp Marian 1981 Anthony amp Fabricius 2000Naumann et al 2011)

Excretion rates were assessed by determining ammonium concentration in seawatersamples (20 ml) that were sterile filtered (02 microm) and kept frozen (minus20 C) untilammonium concentration was determined in 4 replicates per sample through spectroflu-orometric techniques (Holmes et al 1999 protocol B)

Results from calcification respiration and ammonium excretion measurements werenormalized to the coral skeletal surface area (fully covered by coral tissue) to allow forcomparison with other coral species The skeletal surface area (S) of each coral polyp wasdetermined by means of Advanced Geometry (Naumann et al 2009) according to theequation S= π middot (r+R) middota+π middotR2 where r and R represent the basal and apical radius ofeach polyp respectively and a is the apothem measured with a caliper (Rodolfo-Metalpaet al 2006) Finally the ON ratio was calculated for each coral from the results of themeasured oxygen respired and ammonium excreted in atomic equivalents (Yang et al2006 Zonghe et al 2013)

Statistical analysesAll results were expressed as meansplusmn standard error Normal distribution of the residualswas tested using a ShapirondashWilk test performed with the R-language function shapirotestof the R 312 software platform (R Core Team 2014) Homogeneity of variances wastested by the Bartlett test performed with the R-language function bartletttest Differencesin the variation of TA oxygen and ammonium concentration between control and exper-imental chambers were tested by means of a WilcoxonndashMannndashWhitney test performed

Gori et al (2016) PeerJ DOI 107717peerj1606 516

Table 1 Two-way ANOVA for comparison of calcification respiration ammonium excretion ratesand ON ratio among the experimental treatments significant p-values are indicated with one (p-valuelt 005) two (p-value lt 001) or three asterisks (p-value lt 0001)

F p value

Calcification Temperature 858 0019 pCO2 189 0206TemperaturepCO2 044 0524

Respiration Temperature 104 0337pCO2 029 0602TemperaturepCO2 1244 0008

Ammonium excretion Temperature 101 0344pCO2 006 0811TemperaturepCO2 207 0188

ON Temperature 069 0431pCO2 048 0509TemperaturepCO2 794 0023

with the R-language function wilcoxontest Differences among the four experimentalconditions in calcification respiration ammonium excretion and ON ratio were testedby two-way ANOVA with temperature (12 Cndash15 C) and pCO2 (390 ppmndash750 ppm) asfactors performed with the R-language function aov

RESULTSTA changes in incubation chambers (28ndash128 microEq Lminus1 hminus1) were consistently higher(WilcoxonndashMannndashWhitney test U = 48 p = 0004) than changes measured in thecontrol chambers (lt05 microEq Lminus1 hminus1) Regardless of pCO2 level calcification ratesassessed with the TA anomaly technique (Fig 2A) were significantly lower in coralsmaintained at 15 C compared to those maintained at 12 C (ANOVA F = 857 p=0019 Table 1) For each temperature treatment assessed individually calcification didnot significantly differ at either pCO2 level

Oxygen depletion from coral respiration in incubation chambers (53ndash547 micromol Lminus1

hminus1) was significantly higher (WilcoxonndashMannndashWhitney test U = 47 p= 0002) thanoxygen depletion in the control chambers frommicrobial respiration (lt42 micromol Lminus1 hminus1)Respiration rates (Fig 2B) of corals kept under increased temperature and pCO2 weresignificantly lower compared to other treatments (ANOVA F = 1244 p= 0007 Table 1)

Changes in ammonium concentration from coral excretion in incubation chambers(039ndash178 micromol Lminus1 hminus1) were significantly higher (WilcoxonndashMannndashWhitneytest U = 48 p= 0001) than changes in control chambers from microbial activity(lt004 micromol Lminus1 hminus1) Coral excretion rates (Fig 2C) were not significantly differentamong treatments (Table 1)

The ratio of respired oxygen to excreted nitrogen (ON) (Fig 3) in corals kept underincreased temperature and pCO2 was significantly lower than in the other treatments(ANOVA F = 794 p= 0023 Table 1)

Gori et al (2016) PeerJ DOI 107717peerj1606 616

Figure 2 Main physiological processes inDesmophyllum dianthus under the two experimental tem-peratures (12 and 15 C) and the two pCO2 (390 and 750 ppm) (A) Calcification rate (B) respirationrate and (C) ammonium excretion rate as the result of coral nubbins incubation in individual beakers for6 h Values are presented as meansplusmn se normalised to coral skeletal surface area

DISCUSSIONOverall the results of this study show that the CWCD dianthus is more sensitive to changesin temperature than to ocean acidification stress This CWCmaintains itsmetabolismunderelevated pCO2 whereas calcification is significantly reduced under elevated temperaturesFurthermore there is a clear synergistic impact when elevated temperature and pCO2 arecombined resulting in a severe reduction of coral metabolism

D dianthus has the ability to withstand elevated pCO2 (750 ppm) under ambienttemperature (12 C) over sim8 months with no change in calcification respiration and

Gori et al (2016) PeerJ DOI 107717peerj1606 716

Figure 3 Ratio of respired oxygen to excreted nitrogen (ON) ofDesmophyllum dianthus under thetwo experimental temperatures (12 and 15 C) and the two pCO2 levels (390 and 750 ppm) Values arepresented as meansplusmn se normalized to coral skeletal surface area

ammonium excretion rates (Fig 2 and Table 1) This agrees with previous studies onthe same species (Movilla et al 2014b Carreiro-Silva et al 2014 Rodolfo-Metalpa et al2015) and with the general consensus that CWC can physiologically cope with elevatedpCO2 in the mid-term (3ndash12 months Form amp Riebesell 2012 Maier et al 2013a Maieret al 2013b Movilla et al 2014a Hennige et al 2015) This may be due to their ability tobuffer external changes in seawater pH by up-regulating their pH at the site of calcification(McCulloch et al 2012 Anagnostou et al 2012) therefore allowing calcification evenin aragonite-undersaturated seawater (Venn et al 2013) Increased expression of genesinvolved in cellular calcification and energy metabolism may indicate the mechanismsby which D dianthus continues to calcify under elevated pCO2 at rates similar to thoserecorded at ambient pCO2 (Carreiro-Silva et al 2014) Whereas microdensity and porosityof D dianthus skeleton have been shown to be unaffected by increased pCO2 (Movilla etal 2014b) the effects of elevated pCO2 conditions on hidden skeleton microstructureand aragonitic crystals organisation cannot be discounted (eg molecular bond lengthsand orientation see Hennige et al 2015) Such effects would take a long time to becomeevident as reduced skeletal microdensity and porosity due to the very slow growth ratesof D dianthus (Orejas et al 2011 Naumann et al 2011) The experimentally observedphysiological ability of D dianthus to cope with elevated pCO2 is also supported by therecent observation of this CWC in aragonite-undersaturated waters (Thresher et al 2011McCulloch et al 2012 Jantzen et al 2013a Fillinger amp Richter 2013) However there isthe possibility that high food availability in these areas may allow corals to sustain the costof calcification under low pH (Jantzen et al 2013a Fillinger amp Richter 2013)

In contrast to elevated pCO2 elevated temperature alone significantly reducedcalcification in D dianthus (Fig 2B and Table 1) Calcification shows a strong sensitivityto temperature in this CWC species (McCulloch et al 2012) which is able to maintaingrowth under elevated seawater temperatures for a short time (3 months at 175 C

Gori et al (2016) PeerJ DOI 107717peerj1606 816

Naumann Orejas amp Ferrier-Pagegraves 2013) but when exposed to thermal stress for longerperiods (sim8 months at 15 C this study) calcification rates are significantly reducedDecreased calcification in D dianthus under prolonged elevated temperature might belinked to decreased activity in the enzymes involved in calcification (such as carbonicanhydrases Ip Lim amp Lim 1991 Al-Horani Al-Moghrabi amp De Beer 2003 Allemand etal 2004) since enzyme activity is maximal within the thermal range of the speciesand decreases otherwise (Jacques Marshall amp Pilson 1983 Marshall amp Clode 2004Al-Horani 2005) Reported calcification rates by D dianthus have varied widely betweenstudies Rates measured here (126 plusmn 020 micromol CaCO3 cmminus2 dminus1) were in the sameorder of magnitude as the rates reported by Naumann et al (2011) in the Mediterranean(sim384 micromol CaCO3 cmminus2 dminus1) and much lower than those reported by Jantzenet al (2013b) in Chilean fjords (186ndash544 micromol CaCO3 cmminus2 dminus1) Whilst directcomparison with other studies is problematic due to differences in methodology (totalalkalinity vs buoyant weight) or normalization techniques the rates measured hereare consistent with previous results from Mediterranean D dianthus (eg Orejas et al2011 Maier et al 2012 Movilla et al 2014b) and are much higher than rates measuredin D dianthus from Azores (Carreiro-Silva et al 2014) Differences in the quality andquantity of food provided to corals (Mortensen 2001 Jantzen et al 2013b) coral size(Carreiro-Silva et al 2014 Movilla et al 2014b) or intraspecific variability and localadaptation could all contribute to observed variability between studies

The synergistic effects of elevated temperature and pCO2 on calcification respirationand ON ratio observed in this study (Fig 2 and Table 1) show that these stressors interactto controlD dianthusmetabolism causing a far greater effect than increased temperature orpCO2 in isolation (Reynaud et al 2003) Under elevated temperature and pCO2 treatmentrespiration dropped to low values (12 plusmn 07 micromol C cmminus2 dminus1) comparable to thosereported for starved D dianthus (sim15 micromol C cmminus2 dminus1 Naumann et al 2011) or forD dianthus fed only twice a week (134 plusmn 031 micromol C cmminus2 dminus1 Gori et al 2014b)indicating a reduction in the coralrsquos metabolic activity Reduced metabolism is reflectedin the concurrent significant reduction in calcification rates (Fig 2A) Whilst ammoniumexcretion which results from protein and amino acid catabolism (Wright 1995 Talbotamp Lawrence 2002) was not significantly affected by either or both elevated temperatureand pCO2 (consistent with previous studies Carreiro-Silva et al 2014) the combinedeffects of elevated temperature and pCO2 caused a shift in ON from sim30 to sim13 (Fig 3)This highlights a shift from a mixed use of protein and carbohydrate or lipid to a muchless efficient protein-dominated catabolism for energy (Pillai amp Diwan 2002) indicatingmetabolic stress (Zonghe et al 2013) Conversely single stressors caused a slightly increasein ON sim30 to sim50 This is a consequence of increased respiration combined with steadyammonium excretion leading to a shift to a carbohydrate or lipid-dominated metabolism(Sabourin amp Stickle 1981 Uliano et al 2010 Zonghe et al 2013) This is a possible way forthe corals to fulfill increased energy demands needed to maintain cell homeostasis undersingle stressors but this may be insufficient when subjected to multiple stressors

Overall this study shows that the combined effects of increased temperature and pCO2

result in a significant change in D dianthusmetabolism This may represent an immediate

Gori et al (2016) PeerJ DOI 107717peerj1606 916

threat to CWC as their habitats are expected to be exposed to both high temperature eventsand reduced seawater pH with increased frequency in the near future (Roberts amp Cairns2014) Given the major role of feeding on the metabolism of CWC species (Naumannet al 2011) it is also extremely important to understand how coral responses to singleor multiple stressors can be affected by food availability and quality (Dodds et al 2007Thomsen et al 2013 Rodolfo-Metalpa et al 2015) Reduced food availability will limit theallocation of extra-energy to physiological adjustments under stress conditions whichcould further heighten the negative impacts of elevated temperature and pCO2 on coralmetabolism Studies into the combined impact of climate change and changes in foodquantity and quality would provide a more holistic insight into the future of CWC in achanging ocean

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis work was supported by the UK Natural Environment Research Council (grantsNEJ0211211 and NEH0173051 to JMR NEK0090281 to SJH) and the Government ofthe Principality of Monaco JMR LCW and SJH received additional support from Heriot-Watt Universityrsquos Environment and Climate Change theme and the Marine Alliance forScience and Technology Scotland (MASTS) The funders had no role in study design datacollection and analysis decision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsUK Natural Environment Research Council NEJ0211211 NEH0173051NEK0090281Government of the Principality of MonacoHeriot-Watt Universityrsquos Environment and Climate Change themeMarine Alliance for Science and Technology Scotland (MASTS)

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Andrea Gori conceived and designed the experiments performed the experimentsanalyzed the data wrote the paper prepared figures andor tables reviewed drafts of thepaperbull Christine Ferrier-Pagegraves conceived and designed the experiments analyzed the datacontributed reagentsmaterialsanalysis tools wrote the paper reviewed drafts of thepaperbull Sebastian J Hennige and Laura C Wicks wrote the paper reviewed drafts of the paperbull FionaMurray analyzed the data wrote the paper prepared figures andor tables revieweddrafts of the paperbull Ceacutecile Rottier performed the experiments reviewed drafts of the paper

Gori et al (2016) PeerJ DOI 107717peerj1606 1016

bull J Murray Roberts conceived and designed the experiments contributed reagentsmate-rialsanalysis tools wrote the paper reviewed drafts of the paper

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

CITES permit 2012MC7725CITES permit 2012MC7929

Data AvailabilityThe following information was supplied regarding data availability

Raw data is available in the Supplemental Information

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj1606supplemental-information

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calcification in the scleractinian coral Galaxea fascicularis measured with O2 Ca2+

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its relation to photosynthesis and respiration in the scleractinian coral GalaxeafascicularisMarine Biology 142419ndash426 DOI 101007s00227-002-0981-8

Allemand D Ferrier-Pagegraves C Furla P Houlbregraveque F Puverel S Reynaud S Tam-butteacute E Tambutteacute S Zoccola D 2004 Biomineralisation in reef-building coralsfrom molecular mechanisms to environmental control Comptes Rendus Palevol3453ndash467 DOI 101016jcrpv200407011

Anagnostou E Huang KF You CF Sikes EL Sherrell RM 2012 Evaluation of boronisotope ratio as a pH proxy in the deep sea coral Desmophyllum dianthus evidence ofphysiological pH adjustment Earth and Planetary Science Letters 349ndash350251ndash260DOI 101016jepsl201207006

Anthony KRN Fabricius KE 2000 Shifting roles of heterotrophy and autotrophy incoral energy budgets at variable turbidity Journal of Experimental Marine Biology andEcology 252221ndash253 DOI 101016S0022-0981(00)00237-9

Bramanti L Movilla J GuronM Calvo E Gori A Dominguez-Carrioacute C GrinyoacuteJ Lopez-Sanz A Martinez-Quintanilla A Pelejero C Ziveri P Rossi S 2013Detrimental effects of ocean acidification on the economically important Mediter-ranean red coral (Corallium rubrum) Global Change Biology 191897ndash1908DOI 101111gcb12171

Brooke S Ross SW Bane JM SeimHE Young CM 2013 Temperature tolerance ofthe deep-sea coral Lophelia pertusa from the southeastern United States Deep-SeaResearch II 92240ndash248 DOI 101016jdsr2201212001

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Buhl-Mortensen LA Vanreusel AJ Gooday LA Levin I Priede G Buhl-Mortensen PGheerardyn H King NJ Raes M 2010 Biological structures as a source of habitatheterogeneity and biodiversity on the deep ocean marginsMarine Ecology 3121ndash50DOI 101111j1439-0485201000359x

Cairns SD Zibrowius H 1997 Cnidaria Anthozoa azooxanthellate Scleractinia fromthe Philippine and Indonesian regionsMemoirs du Museum National drsquoHistoireNaturelle 17227ndash243

Carreiro-Silva M Cerqueira T Godinho A CaetanoM Santos RS Bettencourt R2014Molecular mechanisms underlying the physiological responses of the cold-water coral Desmophyllum dianthus to ocean acidification Coral Reefs 33465ndash476DOI 101007s00338-014-1129-2

Chan NCS Connolly SR 2012 Sensitivity of coral calcification to ocean acidification ameta-analysis Global Change Biology 19282ndash290 DOI 101111gcb12011

Dodds LA Roberts JM Taylor AC Marubini F 2007Metabolic tolerance of thecold-water coral Lophelia pertusa (Scleractinia) to temperature and dissolvedoxygen change Journal of Experimental Marine Biology and Ecology 349205ndash214DOI 101016jjembe200705013

Edmunds PJ Brown D Moriarty V 2012 Interactive effects of ocean acidification andtemperature on two scleractinian corals from Moorea Freanch Polynesia GlobalChange Biology 182173ndash2183 DOI 101111j1365-2486201202695x

Erez J Reynaud S Silverman J Schneider K Allemand D 2011 Coral calcificationunder ocean acidification and global change In Dubinsky Z Stambler N eds Coralreefs an ecosystem in transition New York Springer 151ndash176

Fillinger L Richter C 2013 Vertical and horizontal distribution of Desmophyllumdianthus in Comau Fjord Chile a cold-water coral thriving at low pH PeerJ 1e194DOI 107717peerj194

Form AU Riebesell U 2012 Acclimation to ocean acidification during long-term CO2

exposure in the cold-water coral Lophelia pertusa Global Change Biology 18843ndash853DOI 101111j1365-2486201102583x

Gattuso JP Frankignoulle M Bourge I Romaine S Buddemeier RW 1998 Effect ofcalcium carbonate saturation of seawater on coral calcification Global and PlanetaryChange 1837ndash46 DOI 101016S0921-8181(98)00035-6

Gori A Grover R Orejas C Sikorski S Ferrier-Pagegraves C 2014b Uptake of dissolved freeamino acids by four cold-water coral species from the Mediterranean Sea Deep-SeaResearch II 9942ndash50 DOI 101016jdsr2201306007

Gori A Reynaud S Orejas C Gili JM Ferrier-Pagegraves C 2014a Physiological perfor-mance of the cold-water coral Dendrophyllia cornigera reveals its preference fortemperate environments Coral Reefs 33665ndash674 DOI 101007s00338-014-1167-9

Guinotte JM Orr JC Cairns SS Freiwald A Morgan L George R 2006Willhuman-induced changes in seawater chemistry alter the distribution of deep-sea scleractinian corals Frontiers in Ecology and Environement 4141ndash146DOI 1018901540-9295(2006)004[0141WHCISC]20CO2

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Hennige SJ Wicks LC Kamenos NA Bakker DCE Findlay HS Dumousseaud CRoberts JM 2014 Short-term metabolic and growth response of the cold-watercoral Lophelia pertusa to ocean acidification Deep-Sea Research II 9927ndash35DOI 101016jdsr2201307005

Hennige SJ Wicks LC Kamenos NA Findlay HS Roberts JM 2015Hidden impacts ofcoral acclimation to ocean acidification Proceedings of the Royal Society B BiologicalSciences 28220150990

Henry LA Roberts JM 2007 Biodiversity and ecological composition of macrobenthoson cold-water coral mounds and adjacent off-mound habitat in the bathyal Porcu-pine Seabight NE Atlantic Deep-Sea Research I 54654ndash672DOI 101016jdsr200701005

Hoegh-Guldberg O 1999 Climate change coral bleaching and the future of the worldrsquoscoral reefsMarine Freshwater Research 50839ndash866 DOI 101071MF99078

Hoegh-Guldberg O Mumby PJ Hooten AJ Steneck RS Greenfield P Gomez EHarvell CD Sale PF Edwards AJ Caldeira K Knowlton N Eakin CM Iglesias-Prieto R Muthiga N Bradbury RH Dubi A Hatziolos ME 2007 Coral reefsunder rapid climate change and ocean acidification Science 3181737ndash1742DOI 101126science1152509

Holmes RM Aminot A Keacuterouel R Hooker BA Peterson BJ 1999 A simple and precisemethod for measuring ammonium in marine and freshwater ecosystems CanadianJournal of Fisheries and Aquatic Sciences 561801ndash1808 DOI 101139f99-128

Ip YK Lim LL Lim RWL 1991 Some properties of calcium-activated adenosine triphos-phate from the hermatypic coral Galaxea fascicularisMarine Biology 111191ndash197DOI 101007BF01319700

Jacques TG Marshall N PilsonMEQ 1983 Experimental ecology of the temperatescleractinian coral Astrangia danae II Effect of temperature light intensity andsymbiosis with zooxanthellae on metabolic rate and calcificationMarine Biology76135ndash148 DOI 101007BF00392730

Jacques TG PilsonMEQ 1980 Experimental Ecology of the temperate scleractiniancoral Astrangia danae I Partition of respiration photosynthesis and calcificationbetween host and symbiontsMarine Biology 60167ndash178 DOI 101007BF00389160

Jantzen C Haumlussermann V Foumlrsterra G Laudien J ArdelanMMaier S Richter C2013a Occurrence of a cold-water coral along natural pH gradients (PatagoniaChile)Marine Biology 1602597ndash2607 DOI 101007s00227-013-2254-0

Jantzen C Laudien J Sokol S Foumlrsterra G Haumlussermann V Kupprat F Richter C2013b In situ short-term growth rates of a cold-water coralMarine and FreshwaterResearch 64631ndash641 DOI 101071MF12200

Jones CG Lawton JH ShachakM 1994 Organisms as ecosystem engineers Oikos69373ndash386 DOI 1023073545850

Langdon C Gattuso JP Andersson A 2010 Measurement of calcification and disso-lution of benthic organisms and communities In Riebesell U Fabry VJ HansonL Gattuso JP eds Guide to best practices for ocean acidification research and datareporting Luxembourg Publications office of the European Union

Gori et al (2016) PeerJ DOI 107717peerj1606 1316

Maier C Bils F Weinbauer MGWatremez P PeckMA Gattuso JP 2013b Respi-ration of Mediterranean cold-water corals is not affected by ocean acidificationas projected for the end of the century Biogeoscience Discuss 107617ndash7640DOI 105194bgd-10-7617-2013

Maier C Schubert A Berzunza SagravenchezMMWeinbauer MGWatremez P Gattuso JP2013a End of the century pCO2 levels do not impact calcification in Mediterraneancold-water corals PLoS ONE 8e62655 DOI 101371journalpone0062655

Maier CWatremez P Taviani MWeinbauer MG Gattuso JP 2012 Calcificationrates and the effect of ocean acidification on Mediterranean cold-water coralsProceedings of the Royal Society B Biological Sciences 2791716ndash1723DOI 101098rspb20111763

Marshall AT Clode P 2004 Calcification rate and the effect of temperature in a zoox-anthellate and an azooxanthellate scleractinian reef coral Coral Reefs 23218ndash224DOI 101007s00338-004-0369-y

McCullochM Trotter J Montagna P Falter J Dunbar R Freiwald A Foumlrsterra GLoacutepez Correa M Maier C Ruumlggeberg A Taviani M 2012 Resilience of cold-water scleractinian corals to ocean acidification boron isotopic systematics of pHand saturation state up-regulation Geochimica et Cosmochimica Acta 8721ndash34DOI 101016jgca201203027

Mcleod E Anthony KRN Andersson A Beeden R Golbuu Y Kleypas J Kroeker KManzello D Salm RV Schuttenberg H Smith JE 2013 Preparing to manage coralreefs for ocean acidification lessons from coral bleaching Frontiers in Ecology and theEnvironment 1120ndash27 DOI 101890110240

Mortensen PB 2001 Aquarium observations on the deep-water coral Lophelia pertusa(L 1758) (Scleractinia) and selected associated invertebrates Ophelia 5483ndash104DOI 10108000785236200110409457

Movilla J Calvo E Pelejero C Coma R Serrano E Fernaacutendez-Vallejo P Ribes M2012 Calcification reduction and recovery in native and non-native Mediterraneancorals in response to ocean acidification Journal of Experimental Marine Biology andEcology 438144ndash153 DOI 101016jjembe201209014

Movilla J Gori A Calvo E Orejas C Loacutepez-Sanz Agrave Domiacutenguez-Carrioacute C Grinyoacute JPelejero C 2014a Resistance of two Mediterranean cold-water coral species to low-pH conditionsWater 659ndash67 DOI 103390w6010059

Movilla J Orejas C Calvo E Gori A Loacutepez-Sanz Agrave Grinyoacute J Domiacutenguez-Carrioacute CPelejero C 2014b Differential response of two Mediterranean cold-water coralspecies to ocean acidification Coral Reefs 33675ndash686DOI 101007s00338-014-1159-9

Muscatine L McCloskey LR Marian RE 1981 Estimating the daily contribution ofcarbon from zooxanthellae to coral animal respiration Limnology and Oceanography26601ndash611 DOI 104319lo19812640601

NaumannMS Niggl W Laforsch C Glaser CWild C 2009 Coral surface areaquantificationmdashevaluation of established methods by comparison with computertomography Coral Reefs 28109ndash117 DOI 101007s00338-008-0459-3

Gori et al (2016) PeerJ DOI 107717peerj1606 1416

NaumannMS Orejas C Ferrier-Pagegraves C 2013High thermal tolerance of two Mediter-ranean cold-water coral species maintained in aquaria Coral Reefs 32749ndash754DOI 101007s00338-013-1011-7

NaumannMS Orejas CWild C Ferrier-Pagegraves C 2011 First evidence for zooplanktonfeeding sustaining key physiological processes in a scleractinian cold-water coralJournal of Experimental Biology 2143570ndash3576 DOI 101242jeb061390

Orejas C Ferrier-Pagegraves C Reynaud S Gori A Beraud E Tsounis G Allemand DGili JM 2011 Long-term growth rate measurements of four Mediterranean coldwater coral species (Madrepora oculata Lophelia pertusa Desmophyllum cristagalliand Dendrophyllia cornigera) maintained in aquariaMarine Ecology Progress Series42957ndash65 DOI 103354meps09104

Pillai BR Diwan AD 2002 Effects of acute salinity stress on oxygen consumption andammonia excretion rates of the marine shrimpMetapenaeus monoceros Journal ofCrustacean Biology 2245ndash52 DOI 10116320021975-99990207

R Core Team 2014 R a language and environment for statistical computing Vienna RFoundation for Statistical Computing Available at httpwwwR-projectorg

Reynaud S Leclercq N Romaine-Lioud S Ferrier-Pagegraves C Jaubert J Gattuso JP2003 Interacting effects of CO2 partial pressure and temperature on photosynthesisand calcification in a scleractinian coral Global Change Biology 91660ndash1668DOI 101046j1365-2486200300678x

Riebesell U Fabry VJ Hansson L Gattuso JP 2010Guide to best practices for oceanacidification research and data reporting Luxembourg Publications Office of theEuropean Union 260 p

Roberts JM Cairns SD 2014 Cold-water corals in a changing ocean Current Opinion inEnvironmental Sustainability 7118ndash112 DOI 101016jcosust201401004

Roberts JMWheeler AJ Freiwald A 2006 Reefs of the deep the biology and geology ofcold-water coral ecosystems Science 312543ndash547 DOI 101126science1119861

Rodolfo-Metalpa R Montagna P Aliani S Borghini M Canese S Hall-Spencer JMFoggo A MilazzoM Taviani M Houlbregraveque F 2015 Calcification is not theAchillesrsquo heel of cold-water corals in an acidifying ocean Global Change Biology212238ndash2248 DOI 101111gcb12867

Rodolfo-Metalpa R Richard C Allemand D Ferrier-Pagegraves C 2006 Growth andphotosynthesis of two Mediterranean corals Cladocora caespitosa and Oculinapatagonica under normal and elevated temperatures Journal of Experimental Biology2094546ndash4556 DOI 101242jeb02550

Sabourin TD StickleWB 1981 Effects of salinity on respiration and nitrogen excretionin two species of echinodermsMarine Biology 6591ndash99 DOI 101007BF00397072

Silverman J Lazar B Cao L Caldeira K Erez J 2009 Coral reefs may start dissolvingwhen atmospheric CO2 doubles Geophysical Research Letters 36L05606

Smith SV Key GS 1975 Carbon dioxide and metabolism in marine environmentsLimnology and Oceanography 20493ndash495 DOI 104319lo19752030493

Talbot TD Lawrence JM 2002 The effect of salinity on respiration excretionregeneration and production in Ophiophragmus filograneus (Echinodermata

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Ophiuroidea) Journal of Experimental Marine Biology and Ecology 2751ndash14DOI 101016S0022-0981(02)00208-3

Thomsen J Casties I Pansch C Koumlrtzinger A Melzner F 2013 Food availabilityoutweighs ocean acidification effects in juvenileMytilus edulis laboratory and fieldexperiments Global Change Biology 191017ndash1027 DOI 101111gcb12109

Thresher RE Tilbrook BD Fallon S Wilson NC Adkins J 2011 Effects of chroniclow carbonate saturation levels on the distribution growth and skeletal chemistryof deep-sea corals and other seamount megabenthosMarine Ecology Progress Series44287ndash99 DOI 103354meps09400

Uliano E Cataldi M Carella F Migliaccio O Iaccarino D Agnisola C 2010 Effectsof acute changes in salinity and temperature on routine metabolism and nitrogenexcretion in gambusia (Gambusia affinis) and zebrafish (Danio rerio) ComparativeBiochemistry and Physiology Part A 157283ndash290 DOI 101016jcbpa201007019

Venn AA Tambutte E HolcombM Laurent J Allemand D Tambutte S 2013 Impactof seawater acidification on pH at the tissue-skeleton interface and calcification inreef corals Proceedings of the National Academy of Sciences of the United States ofAmerica 1101634ndash1639 DOI 101073pnas1216153110

Wright PA 1995 Nitrogen excretion three end products many physiological rolesJournal of Experimental Biology 198273ndash281

Yang H Zhou Y Zhang T Yuan X Li X Liu Y Zhang F 2006Metabolic char-acteristics of sea cucumber Apostichopus japonicus (Selenka) during aesti-vation Journal of Experimental Marine Biology and Ecology 330505ndash510DOI 101016jjembe200509010

Zonghe Y Zhanhui Q Chaoqun HWenguang L Huang H 2013 Effects of salin-ity on ingestion oxygen consumption and ammonium excretion rates of thesea cucumber Holothuria leucospilota Aquaculture Research 441760ndash1767DOI 101111j1365-2109201203182x

Gori et al (2016) PeerJ DOI 107717peerj1606 1616